This application is a U.S. national application of international application serial No. PCT/EP98/02238 filed Apr. 16, 1998, which claims priority to German serial No. 197 17 864.2 filed Apr. 23, 1997.
The invention pertains to variants of human recombinant beta-interferon with improved solubility.
Beta-interferon is a regulatory protein which leads to activation of genes by binding receptors. As a result, antiviral, antiproliferative and further biological activities are mediated in the cell.
The inteferons, as is also the case with the interleukins, belong to the class of cytokines and are subdivided into different classes:
Type I interferon (alpha, beta, omega, tau) and Type II (gamma)
Human beta-interferon is a protein with a molecular weight of 22 kDa and 166 amino acid residues. It is formed primarily in fibroblasts during attack by a virus and possesses antiviral, antiproliferative and further biological activities. The amino acid sequence of human beta-interferon was first published by Taniguchi et al. (1980), Gene Ed. 10, pages 11 through 15, and is illustrated in FIG. 1.
Beta-interferon, which is produced from bacterial cells or mammalian cells by genetic engineering, is being used successfully in the treatment of multiple sclerosis, a previously incurable disease in a large group of patients. However, the very high hydrophobicity of the protein, which causes very poor solubility of recombinant human beta-interferon, proves to be problematical for the production and processing of recombinant human beta-interferon.
The problem for the present invention is to make available variants of recombinant human beta-interferon whose solubility is improved in polar media, such as e.g. aqueous liquids. In addition an objective of this invention is to indicate processes for manufacturing and possibilities for using variants of recombinant human beta-interferon with higher solubility in polar media such as aqueous liquids.
This problem is solved by the recombinant human beta-interferon in accordance with claim 1, its use in accordance with claim 5 and its manufacture in accordance with claim 6 or 7.
In accordance with claim 1, at least one of the following ten hydrophobic amino acids in known human beta-interferon is exchanged for a hydrophilic amino acid: Leu 5, Phe 8, Phe 15, Leu 47, Phe 50, Leu 106, Phe 111, Leu 120 or Phe 156 (SED ID No.2). Thus the invention pertains to individual mutations as well as to all the possible combinations of these individual amino acid exchanges.
The designated amino acids are essentially located on the surface of human beta-interferon and they take up a relatively large proportion of the surface there. The exchange of these amino acids therefore leads to more than a proportionately large improvement in the hydrophilic character of the surface of recombinant human beta-interferon and it therefore increases the solubility of this protein in polar media, such as e.g. aqueous liquids. As a result of its increased hydrophilicity, the recombinant human beta-interferon in accordance with the invention is considerably simpler to handle in production as well as during its processing to give an active substance.
The production of the variants of recombinant human beta-interferon in accordance with the invention takes place in a generally known, conventional way with the help of microorganisms, e.g. in an Escherichia coli culture which has been provided with the gene for one of the proteins in accordance with the invention. The production of these microorganisms, which have been changed by means of genetic engineering, also takes place in a generally known way with the help of classical genetic engineering mutagenesis procedures for the exchange of the corresponding amino acids for hydrophilic amino acids and their synthesis will therefore be dispensed with at this juncture.
The proteins in accordance with the invention find use for the manufacture of medicinal drugs, e.g. for combatting multiple sclerosis, as well as fine chemicals for in vitro experiments or for measurements of interferon levels. The improved hydrophilicity of these proteins thereby simplifies their manufacture, transportation, storage and application in the form of a medicinal drug or fine chemical.
Advantageous further developments of the proteins in accordance with the invention are given in the dependent claims.
Exchange for the amino acids serine, tyrosine and threonine is especially advantageous and, with one hydroxy group each, these are especially hydrophilic.
As a result of its small size, serine is especially suitable for exchange since especially slight steric changes in the protein are associated with it.
The amino acid sequence of native recombinant human beta-interferon, in which the abovementioned ten amino acids are electively exchanged for serine, is illustrated in FIG. 2. These exchangeable amino acids are represented by Xaa. If these amino acids are exchanged, then the hydrophilicity of the surface of recombinant human beta-interferon is very much improved whereas only slight impairment arises in terms of the functionality and the efficacy of human recombinant beta-interferon.
An especially advantageous further development is illustrated in FIG. 3 in which all of the abovementioned ten amino acids have been exchanged here for serine so that an especially marked increase in the hydrophilicity of the surface of recombinant human beta-interferon results.